Prof. Dr. Dr. h.c. Hartmut Michel, Director
As outlined below, it is the aim of the department to understand how membrane proteins function and to determine their precise mechanism of action.
Basic features of biological membranes and the function of membrane proteins
Membranes surround biological cells and divide the cells of higher organisms into various compartments. Biological membranes are composed of lipids, which form a bilayer, and of membrane proteins which are incorporated into the lipid bilayer. Lipid bilayers are impermeable to ions and to polar substances. As a consequence of this property ion gradients and electric voltages (“membrane potentials”) can be formed across membranes. Membrane proteins are required to enable the specific passage or transport of selected substances across membranes. Enabling passage and transport is therefore one of the most important functions of membrane proteins. Cells have to communicate, receive signals and to sense their environment. Many membrane proteins are sensors and receptors, the signal is often received at the outer face of the membrane and transduced across the membrane. Membrane proteins are central components of biological energy conversion. In photosynthesis and cellular respiration the transport of electrons and protons across membranes is the primary step of energy conversion. The resulting membrane potentials and ion gradients can be used to drive the synthesis of adenosine-5’-triphosphate (ATP), the uptake of nutrients, the export of waste products and proteins as well to drive the flagella motors. Finally, some membrane proteins are enzymes, in particular, when the substrates and/or products are hydrophobic.
Summarizing, the functions of membrane proteins can be classified as follows:
(i) Passage and transport. Passage of substances and ions (“passive transport”) is catalyzed by channels, pores and permeases. “Primary active transport” is driven by ATP or other energy-rich substances. In “secondary active transport” the downhill flow of an ion (normally a sodium ion or a proton) is coupled to the transport of a substrate.
(ii) Signal reception and transduction. The most important receptors are the G-protein coupled receptors. Binding of an agonist to the receptor leads to an activation of trimeric G-proteins and the start of a signal transduction cascade.
(iii) Biological energy conversion. The prime examples are the respiratory chains of mitochondria and bacteria, where oxidation of substrates is coupled to the transfer of electric charges across the mitochondrial or bacterial membrane. In photosynthesis the photosynthetic reaction centres use the energy of light for the primary charge separation and transport of electrons across the photosynthetic membrane.
(iv) Enzymes, preferentially for hydrophobic substrates and/or products.
The functional classification of a membrane protein may not be unique, because a receptor might be a channel protein working by opening or closing a channel upon interaction with its ligand.
Aims of the department and methods used
The aim of the department is to understand how membrane proteins function and to determine their precise mechanism of action. Such an understanding is only possible on the basis of an accurately known atomic structure. However, obtaining such a structural knowledge is the bottleneck, because X-ray crystallography, by far the most suited method to determine atomic structures of membrane proteins, requires well ordered crystals and these are difficult to generate. The department has an outstanding proven track record in crystallizing membrane proteins and determining their structure. A list of the membrane protein structures determined in this department can be found at the end of this page.
Once the structure determination of a membrane protein is finished, strong efforts are put into understanding function and mechanism by combined approaches using genetic methods, specific labelling and biophysical techniques. Such work includes site-specific mutagenesis and an enzymatic and structural characterization of the resulting variants. Various spectroscopic methods, like nuclear magnetic resonance (nmr) spectroscopy, Fourier Transform infrared (FTIR) spectroscopy and electron paramagnetic resonance spectroscopy are used to investigate structural changes connected to the membrane protein’s reaction cycle. These spectroscopic experiments are mainly done in collaborations with our colleagues from Frankfurt University (nmr: Profs. Glaubitz, Doetsch and Schwalbe, epr: Prof. Prisner, FTIR: Prof. Mäntele). For electrophysiological investigations we use the expertise of our colleagues from the institute’s department of Biophysical Chemistry. We measure electric currents and voltages accompanying the action of membrane proteins incorporated into lipid bilayers.
The usage of methods from computational biophysics, in particular electrostatic calculations and molecular dynamics simulations, is of invaluable help to better understand the action of membrane proteins, and to make predictions about the role of individual amino acids, which then can be tested experimentally.
Members of the department work on a number of membrane proteins from various functional classes. Among the transport proteins our emphasis lies on secondary active transporters. Here we use a structural genomics type of approach using the hyperthermophilic bacterium Aquifex aeolicus, the mesophilic bacterium Salmonella typhimurium and the archaeon Pyrococcus furiosus as source organisms. We could determine the structure of the NhaA antiporter from Escherichia coli, such a secondary active transporter. Now we aim to obtain the structure of its active form at high pH and shed light into the mechanism of transport.
We used to work on all complexes of the mitochondrial respiratory chain. We have determined structures of respiratory complexes II, III and IV. With the departures of Roy C. Lancaster and Carola Hunte and their groups work on complexes II (succinate dehydrogenases, fumarate reductases) and III (cytochrome bc1 complex) has been discontinued at the institute. Work on complex IV (cytochrome c oxidase) and other aerobic terminal oxidases constitutes a major focus. In a structural proteomics project on Aquifex aeolicus we isolate and crystallize as many membrane proteins and membrane protein complexes as possible. These include the respiratory chain complex I (NADH dehydrogenase) and complex V (ATP-synthase).
Work on G-protein coupled receptors (GPCRs) is ongoing. The emphasis has been shifted to the generation and characterization of functional complexes. We express the GPCRs and their interaction partners in Pichia pastoris, in insect cells, and in mammalian cells using the Semliki Forest Virus expression system.
Ulrich Ermler’s research group studies enzymes of microbial degradation processes, in particular of the methanogenic and the methanotrophic pathway, of dissimilatory sulphate reduction and of degradation of aromatic compounds. The major method used is X-ray crystallography.
Computational methods are employed for analysing structures of membrane proteins. Electrostatic interactions are of particular importance for membrane proteins because of the low dielectric environment in membranes. Molecular dynamics simulations and modelling are other methods used to identify the role of individual amino acids, which then can be tested experimentally.
More than other institutions the department is devoted to the development of methods to investigate membrane proteins. In the past the department head has suggested the currently used strategies to crystallize membrane proteins either within their detergent micelles (relying on crystal contacts to be made primarily by amino acid residues of the extramembranous polar surface) or as stacks of two-dimensional crystals of membrane proteins. The idea to co-crystallize membrane proteins with fragments of monoclonal antibodies originated in the department, and its usefulness has been successfully demonstrated first with the cytochrome c oxidase from the soil bacterium Paracoccus denitrificans. Another “first” has been the employment of the methylotrophic yeast Pichia pastoris for the production of recombinant membrane proteins. At the moment we explore various systems for the cell-free expression of membrane proteins. Because membrane proteins function as lipid-protein complexes rather than as simple membrane proteins we put efforts into the analysis of the lipid contents of our isolated membrane proteins and of our membrane protein crystals using our mass spectrometry facility. It is also employed for the identification of the crystallized membrane proteins and their potential modifications. In parallel we try to develop novel detergents for solubilization and crystallization of membrane proteins.
The department hosts core centre G of the European initiative INSTRUCT (INtegrated STRUCTural Biology infrastructure for Europe) as an intended part of the ESFRI (European Strategy Forum on Research Infrastructures). Core centre G is focused on membrane proteins and provides highly automated platforms for membrane protein crystallization, a most powerful in-house X-ray generator coupled to a CCD detector for testing crystals, and a differential scanning calorimeter for determining the stability of membrane proteins. Our mass spectrometry facility mentioned above is part of core centre G.
The department provided a list of the membrane proteins of known structures with an emphasis on the crystallization conditions. Because of lack of man power it is no longer updated. However, since it is the only comprehensive data base providing information about the detergents and precipitants used successfully, access is still enabled.
Membrane protein structures determined in the department
1prc The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis (now called Blastochloris viridis), first atomic structure of a membrane protein. Hartmut Michel received the 1988 Nobel Prize in Chemistry together with Johann Deisenhofer and Robert Huber for the structure determination.
1pcr The photosynthetic reaction centre from the purple bacterium Rhodobacter sphaeroides. This structure was the first real structure of the Rhodobacter sphaeroides reaction centre. Previous structures suffered from insufficient data leading to an over-refinement and an unjustified deviation from the parent Rhodopseudomonas viridis reaction centre structure used for molecular replacement.
1qle, 1ar1, 3hb3 The cytochrome c oxidase from the soil bacterium Paracoccus denitrificans complexed with an antibody Fv fragment. This was the first complete structure of a respiratory chain complex. 1ar1, 3hb3 represent refined structures of the active two-subunit core complex at higher resolution.
3mk7 The structure of cbb3 cytochrome oxidase from Pseudomonas stutzeri.
1lgh The light-harvesting complex II (B800-850) from Rhodospirillum molischianum (now called Phaeospirillum molischianum).
1qlb Respiratory complex II-like fumarate reductase from Wolinella succinogenes
1ezv Respiratory complex III (cytochrome bc1 complex) from the yeast complexed with an antibody Fv fragment
3cx5 Respiratory complex III (cytochrome bc1 complex) from the yeast complexed with an antibody Fv fragment and its substrate cytochrome c
1zcd The sodium ion/ proton exchanger NhaA from Escherichia coli, down regulated at pH 4.
Prof. Dr. Dr. h.c. Hartmut Michel, Managing Director
Department of Molecular Membrane Biology
Secretary: S. McCormack
Phone: +49 (0) 69 6303-1001
Fax: +49 (0) 69 6303-1002